Systems and methods for high throughput analysis of conformation in biological entities

ABSTRACT

Methods, devices, and systems are disclosed for performing high throughput analysis of conformational change in biological molecules or other biological entities using surface-selective nonlinear optical detection techniques.

CROSS-REFERENCE

This application is a Continuation Application of U.S. application Ser.No. 15/965,577, filed Apr. 27, 2018, which is a Continuation Applicationof U.S. application Ser. No. 14/754,465, filed Jun. 29, 2015, whichclaims the benefit of U.S. Provisional Application No. 62/019,285, filedJun. 30, 2014, each of which is incorporated herein by reference in itsentirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Grant Number IIP-1256619 from the National Science Foundation.

BACKGROUND OF THE INVENTION

Over the past two decades, the advent of high throughput experimentationhas transformed the way life science and biomedical research is carriedout. A convergence of technologies in fields such as geneticengineering, organic chemistry, materials science, microfabrication andmicroelectronics has led to new technology platforms (e.g. microarraytechnologies, microfluidic devices and systems, bead-based combinatorialcompound libraries and assay systems, and microplate-based assaysystems) to address applications ranging from high throughput screeningof compound libraries for drug discovery to rapid whole genomesequencing.

Examples of high throughput screening systems for drug discovery includemicrofluidics-based platform technologies for running continuous-flowassays (e.g. receptor-ligand binding assays and cell-based assays foridentifying receptor agonists), and microplate-based systems in whichbinding reactions, enzymatic reactions, or cell-based assays are run ina microwell plate format, and automated liquid-dispensing stations andplate-handling robotics provide for automated sample preparation, assay,and detection steps. The majority of existing high throughput platformtechnologies for drug discovery utilize fluorescence-based opticaldetection. Although fluorescence techniques provide for very highdetection sensitivity, and are generally much more environmentallyfriendly than the more traditional, radioisotope-based approaches thatpredominated in biological assay methodologies of two decades ago, thereare a number of drawbacks to the use of fluorescence. Examples include:(i) the requirement for sophisticated light sources, detectors, andoptical systems, the performance of which are often sensitive tomisalignment or instrumental drift, and (ii) photo-bleaching phenomena,which may result in degradation of signal over time in samples subjectedto repeat measurements.

Another, more serious limitation of existing high throughput screeningtechnologies stems from the growing awareness that a number of potentialtherapeutic targets, e.g. potential cancer therapeutic targets, that areattractive targets from a biological perspective are intractable(“undruggable”) from a chemical standpoint because they are generallynot amenable to conventional drug discovery approaches. These proteintargets typically possess a relatively large contact area wheninteracting with other proteins (i.e. through protein-proteininteractions) or due to the fact that they possess a ligand that bindswith extremely high affinity to the active site of the protein. Ineither case, finding a conventional small molecule or biologic (protein)drug candidate that will block the interaction (i.e. interfere withand/or obscure the large contact area in the case of protein-proteininteractions, or displace the high affinity ligand) is extremelydifficult. Allosteric modulators for such “undruggable” targets offer anattractive therapeutic solution. By definition, allosteric moleculesbind to a site other than a protein's active site, thereby changing theprotein's conformation with a concomitant functional effect (e.g.activation of a receptor). Allosteric modulation of target proteins hasthe added benefit of not having to rely on inhibition or competitionwith the binding of the natural ligand to the protein, which can resultin unintended clinical side effects. However, it has been difficult toidentify allosteric modulators using currently available conventionaltechniques. For example, structural information obtained from X-raycrystallography or NMR methods is often of limited value for drugdiscovery purposes due to low throughput, low sensitivity, thenon-physiological conditions utilized, the size of the protein amenableto the technique, and many other factors. What is needed, therefore arehigh throughput techniques for screening collections of candidatecompounds to rapidly identify agents capable of, for example, allostericmodulation of the target protein's conformation.

As described more fully below, second harmonic generation (SHG) is anonlinear optical process which may be configured as surface-selectivedetection technique that enables detection of conformational change inproteins and other biological targets (as described previously, forexample, in U.S. Pat. No. 6,953,694, and U.S. patent application Ser.No. 13/838,491). In order to deploy SHG-based detection ofconformational change in a high throughput format, it may beadvantageous to design novel mechanisms for rapid, precise, andinterchangeable positioning of substrates (comprising the biologicaltargets to be analyzed) with respect to the optical system used todeliver excitation light, which at the same time ensure that efficientoptical coupling between the excitation light and the substrate surfaceis maintained. One preferred format for high throughput opticalinterrogation of biological samples is the glass-bottomed microwellplate.

The systems and methods disclosed herein provide mechanisms for couplingthe high intensity excitation light required for SHG and other nonlinearoptical techniques to a substrate, e.g. the glass substrate in aglass-bottomed microwell plate, by means of total internal reflection ina manner that is compatible with the requirements for a high throughputanalysis system.

SUMMARY OF THE INVENTION

Disclosed herein are methods for determining interactions betweenbiological entities and test entities, comprising: (a) contacting eachof at least four biological entities with at least one test entity; (b)illuminating each of the at least four biological entities with one ormore excitation light beams using a surface selective optical technique;and (c) determining whether or not a conformational change was inducedin each of the at least four biological entities through contact withthe at least one test entity; wherein determinations of conformationalchange are performed at an average rate of at least 10 test entitiestested per hour. In some embodiments, the determinations ofconformational change are performed at an average rate of at least 100test entities tested per hour.

In some embodiments, the at least four biological entities are the same.In some embodiments, the at least four biological entities aredifferent. In some embodiments, the biological entities are selectedfrom the group consisting of cells, proteins, peptides, receptors,enzymes, antibodies, DNA, RNA, oligonucleotides, small molecules, andcarbohydrates, or any combination thereof. In some embodiments, thebiological entities are drug targets or portions thereof.

In some embodiments, each of the at least four biological entities aresituated in a different discrete region on a substrate. In someembodiments, each discrete region comprises an area of up to about 100mm² on a substrate surface. In some embodiments, each discrete regioncomprises a supported lipid bilayer. In some embodiments, the biologicalentities are tethered to or embedded within the supported lipid bilayer.

In some embodiments, the at least one test entity is selected from thegroup consisting of cells, proteins, peptides, receptors, enzymes,antibodies, DNA, RNA, oligonucleotides, small molecules, andcarbohydrates, or any combination thereof. In some embodiments, the testentity is a drug candidate or portion thereof.

In some embodiments, the contacting step occurs in solution andcomprises utilizing a pre-programmed fluid dispensing unit to dispensethe at least one test entity. In some embodiments, the contacting stepcomprises contacting each of the at least four biological entities witha different test entity. In some embodiments, the contacting stepcomprises serially contacting each of the at least four biologicalentities with at least 100 different test entities. In some embodiments,the contacting step comprises serially contacting each of the at leastfour biological entities with at least 10,000 different test entities.

In some embodiments, the one or more excitation light beams are providedby one or more lasers. In some embodiments, the surface selectiveoptical detection technique comprises the use of total internalreflection of at least one excitation light beam from a planar substratesurface.

In some embodiments, the determining step further comprises analyzing anon-linear optical signal. In some embodiments, the non-linear opticalsignal is selected from the group consisting of second harmonic light,sum frequency light, and difference frequency light.

In some embodiments, the methods further comprise moving said substraterelative to the position of one or more external sources of the one ormore excitation light beams. In some embodiments, each discrete regionis optically coupled with an entrance prism and a different exit prismthat are both integrated with a bottom surface of the substrate. In someembodiments, the illuminating step comprises directing incidentexcitation light onto an entrance prism positioned adjacent to, but notdirectly below, each of the discrete regions. In some embodiments, themethods further comprise collecting non-linear optical signals generatedat each of the discrete regions upon illumination using an exit prismpositioned adjacent to, but not directly below, each of the discreteregions, and directing said non-linear optical signals to a detector. Insome embodiments, the methods further comprise repeating theilluminating and determining steps a plurality of times after saidcontacting step, thereby determining conformational changes in each ofthe at least four biological entities as a function of time.

Also disclosed herein are devices comprising: (a) a substrate, thesubstrate comprising: (i) an M×N array of discrete regions formed on asurface of the substrate, wherein M is the number of rows of discreteregions and N is the number of columns of discrete regions in the array,and each discrete region is configured for containing a biologicalentity, and (ii) an R×S array of prisms integrated with the substrateand optically coupled to the discrete regions, wherein R is the numberof rows of prisms and S is the number of columns of prisms in the array;wherein R=M+2 and S=N, or R=M and S=N+2.

In some embodiments, each of the discrete regions is optically coupledwith at least one input prism and at least one output prism, and whereinthe input prism and the output prism are spatially distinct. In someembodiments, M=8 and N=12. In some embodiments, M=16 and N=24. In someembodiments, M=32 and N=48. In some embodiments, M is greater than 4 andN is greater than 4. In some embodiments, each discrete region comprisesa supported lipid bilayer or is configured to facilitate the formationof a supported lipid bilayer. In some embodiments, the devices furthercomprise a well-forming component bonded to a top surface of thesubstrate in order to isolate each discrete region in a separate well.In some embodiments, each of the discrete regions comprises an area ofup to about 100 mm². In some embodiments, each discrete region or wellis located directly above a single prism of the array of prismsintegrated with the substrate. In some embodiments, the substrate iscomposed of glass, fused-silica, or plastic.

Disclosed herein are injection molding processes for fabricating a prismarray part from a plastic, the process comprising the use of two or moremold ejection devices to apply uniform pressure to the prism array partduring a mold release step.

In some embodiments, the plastic is selected from the group consistingof cyclic olefin copolymer (COC), cyclic olefin polymer (COP), andacrylic. In some embodiments, the two or more mold ejection devicesimpact the prism array part only in regions where the opticalperformance of the part is non-critical. In some embodiments, the two ormore mold ejection devices comprise an array of m×n blade-like ejectorfeatures. In some embodiments, m is greater than 2 and less than 20. Insome embodiments, n is greater than 2 and less than 20.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A provides a schematic illustration of the energy level diagramsfor fluorescence (an absorption process).

FIG. 1B provides a schematic illustration of the energy level diagramsfor second harmonic generation (a two photon scattering process).

FIG. 2 provides a schematic illustration of a conformational change in aprotein (labeled with a nonlinear-active tag) which is induced bybinding of a ligand, and its impact on the distance and/or orientationof a nonlinear-active label relative to an optical interface to whichthe protein is attached.

FIGS. 3A-D show data that demonstrate the detection of spermine- orspermidine-induced conformational changes in alpha-synuclein usingsecond harmonic light generation. FIG. 3A shows the SHG response in realtime upon exposure of alpha-synuclein to 5 mM spermine. The arrowdenotes spermine addition. The change in SHG intensity is normalized tothe value just prior to injection. FIG. 3B shows the dose response curve(plotted on a log scale) for spermine-induced conformational change inalpha-synuclein as measured by SHG (the change in SHG concentration isquantified as percent shift). FIG. 3C shows the SHG response in realtime upon exposure of alpha-synuclein to 3 mM spermidine. The arrowdenotes spermidine addition. The change in SHG intensity is normalizedto the value just prior to injection. FIG. 3D shows the dose responsecurve (plotted on a log scale) for spermidine-induced conformationalchange in alpha-synuclein as measured by SHG (the change in SHGconcentration is quantified as percent shift). Error bars=SEM. N=3.

FIG. 4 illustrates one example of the system architecture for a highthroughput analysis system for determining conformational change inbiological molecules or other biological entities based on nonlinearoptical detection.

FIG. 5 shows a schematic for one example of an optical setup used foranalysis of conformational change in biological molecules usingnonlinear optical detection.

FIG. 6 shows a photograph of an optical setup used for analysis ofconformational change in biological molecules using nonlinear opticaldetection.

FIG. 7 shows a schematic illustration depicting the use of a prism todirect excitation light at an appropriate incident angle such that theexcitation light undergoes total internal reflection at the top surfaceof a substrate. The two dashed lines to the right of the prism indicatethe optical path of the reflected excitation light and the nonlinearoptical signal generated at the substrate surface when nonlinear-activespecies are tethered to the surface. The substrate is optionallyconnected to the actuator of an X-Y translation stage for re-positioningbetween measurements. The curved lines between the top surface of theprism and the lower surface of the substrate indicate the presence athin layer (not to scale) of index-matching fluid used to ensure highoptical coupling efficiency between the prism and substrate.

FIGS. 8A-C show different views of one exemplary design concept for asystem that uses a continuously recirculating flow of index-matchingfluid to provide high optical coupling efficiency between the prism(attached to the optical instrument in this example) and the substrate(configured as the transparent bottom of a microwell plate in thisexample). The substrate (microwell plate) is free to translate relativeto the prism while a continuous flow of index-matching fluid provided bythe indicated fluid channels ensures good optical coupling of excitationlight with the substrate. FIG. 8A: top-front axonometric view. FIG. 8B:top-rear axonometric view. FIG. 8C: bottom-front axonometric view.

FIG. 9 shows a schematic illustration depicting the use of a layer ofindex-matching elastomeric material attached or adjacent to the lowersurface of a transparent substrate (configured in a microwell plateformat in this example) to ensure high optical coupling efficiencybetween a prism and the upper surface of the substrate. In someembodiments of this approach, the upper surface of the prism is slightlydomed to focus the compression force when bringing the microwell plateand prism into contact, thereby reducing or eliminating the formation ofair gaps between the prism and elastomeric material.

FIGS. 10A-B illustrate a microwell plate with integrated prism array forproviding good optical coupling of the excitation light to the topsurface of the substrate. In this approach, the prism indicated in theschematic illustrations of FIGS. 4, 5, 7, and 9 are replaced by theprism array attached to the underside of the substrate. FIG. 10A: topaxonometric view. FIG. 10B: bottom axonometric view.

FIGS. 10C-D show exploded views of the microwell plate device shown inFIGS. 10A-B. FIG. 10C: top axonometric view. FIG. 10D: bottomaxonometric view.

FIG. 11 illustrates the incident and exit light paths for coupling theexcitation light to the substrate surface via total internal reflectionusing the design concept illustrated in FIGS. 10A-B.

FIG. 12 shows a photograph of a prototype for the prism array designconcept illustrated in FIGS. 10A-B.

FIGS. 13A-C show one example of a prism array design according to thepresent disclosure. FIG. 13A: top view. FIG. 13B: front view. FIG. 13C:right side view.

FIG. 14 shows a crossed-polarizer image of a prism array fabricatedusing a first mold design and first injection molding process. Note thehigh level of stress-induced birefringence observed.

FIG. 15 shows data for the SHG signal intensity measured at differentpositions in a microwell plate device incorporating the prism arraydesign illustrated in FIGS. 13A-C and FIG. 14. SHG signal intensity wasmeasured for different rows (different traces) as a function of wellcolumn number.

FIGS. 16A-C show an example of an improved prism array design accordingto the present disclosure. FIG. 16A: top view. FIG. 16B: front view.FIG. 16C: right side view.

FIG. 17 shows a crossed-polarizer image of a prism array fabricatedusing an improved mold design and optimized injection molding process.Note that the level of stress-induced birefringence observed issignificantly reduced compared to that shown in FIG. 14.

FIG. 18 shows data for the SHG signal intensity measured at differentpositions in a microwell plate device incorporating the prism arraydesign illustrated in FIGS. 16A-C and FIG. 17. SHG signal intensity wasmeasured for different rows (different traces) as a function of wellcolumn number. Note that the level of SHG signal intensity is higher andmore uniform across the microwell plate compared to that shown in FIG.15.

FIG. 19 shows a cut-away version of the mold tool used to fabricate theprism array part illustrated in FIGS. 16A-C.

FIG. 20 illustrates the array of blade-like ejection features used inthe mold tool for providing uniform pressure on the prism array partduring release from the mold.

FIG. 21 illustrates a computer system that may be configured to controlthe operation of the systems disclosed herein.

FIG. 22 is a block diagram illustrating a first example architecture ofa computer system that can be used in connection with exampleembodiments of the present invention.

FIG. 23 is a diagram showing one embodiment of a network with aplurality of computer systems, a plurality of cell phones and personaldata assistants, and Network Attached Storage (NAS).

FIG. 24 is a block diagram of a multiprocessor computer system using ashared virtual address memory space in accordance with an exampleembodiment.

DETAILED DESCRIPTION OF THE INVENTION

The systems and methods disclosed herein relate to high throughputanalysis of conformation in biological entities. In addition, thesystems and methods described are equally suitable for high throughputanalysis of orientation or conformational change. In some aspects of thepresent disclosure, systems and methods are described for determiningorientation, conformation, or changes in orientation or conformation ofbiological entities in response to contacting the biological entitieswith one or more test entities. As used herein, determining orientation,conformation, or changes thereof may involve measurement of a nonlinearoptical signal which is related to and/or proportional to the averageorientation of a nonlinear-active label or tag. As used herein, “highthroughput” refers to the ability to perform rapid analysis ofconformation for a large plurality of biological entities optionallycontacted with one or more test entities, or to the ability to performrapid analysis of conformation for one or more biological entitiesoptionally contacted with a large plurality of test entities, or to anycombination of these two modalities. In general, the systems and methodsdisclosed rely on the use of second harmonic generation (SHG) or relatednonlinear optical techniques for detection of orientation, conformation,or conformational change, as described previously, for example, in U.S.Pat. No. 6,953,694, and U.S. patent application Ser. No. 13/838,491.

Detection of Conformation Using Second Harmonic Generation

Second harmonic generation, in contrast to the more widely usedfluorescence-based techniques, is a nonlinear optical process, in whichtwo photons of the same excitation wavelength or frequency interact witha nonlinear material and are re-emitted as a single photon having twicethe energy, i.e. twice the frequency and half the wavelength, of theexcitation photons (FIGS. 1A-1B). Second harmonic generation only occursin nonlinear materials lacking inversion symmetry (i.e. innon-centrosymmetric materials) and requires a high intensity excitationlight source. It is a special case of sum frequency generation and isrelated to other nonlinear optical phenomena such as differencefrequency generation.

Second harmonic generation and other nonlinear optical techniques can beconfigured as surface-selective detection techniques because of theirdependence on the orientation of the nonlinear-active species. Tetheringof the nonlinear-active species to a surface, for example, can instillan overall degree of orientation that is absent when molecules are freeto undergo rotational diffusion in solution. An equation commonly usedto model the orientation-dependence of nonlinear-active species at aninterface is:

χ⁽²⁾ =N _(s)<α⁽²⁾>

where χ⁽²⁾ is the nonlinear susceptibility, N_(s) is the total number ofnonlinear-active molecules per unit area at the interface, and <α⁽²⁾> isthe average orientation of the nonlinear hyperpolarizability (α⁽²⁾) ofthese molecules. The intensity of SHG is proportional to the square ofthe nonlinear susceptibility, and is thus dependent on both the numberof oriented nonlinear-active species at the interface and to changes intheir average orientation.

Second harmonic generation and other nonlinear optical techniques may berendered additionally surface selective through the use of totalinternal reflection as the mode for delivery of the excitation light tothe optical interface on which nonlinear-active species have beenimmobilized. Total internal reflection of the incident excitation lightcreates an “evanescent wave” at the interface, which may be used toselectively excite only nonlinear-active labels that are in closeproximity to the surface, i.e. within the spatial decay distance of theevanescent wave, which is typically on the order of tens of nanometers.Total internal reflection may also be used to excite fluorescence in asurface-selective manner, for example to excite a fluorescence donorattached to the optical interface, which then transfers energy to asuitable acceptor molecule via a fluorescence resonance energy transfer(FRET) mechanism. In the present disclosure, the evanescent wavegenerated by means of total internal reflection of the excitation lightis preferentially used to excite a nonlinear-active label or molecule.The the efficiency of exciting nonlinear active species will dependstrongly on both their average orientation and on their proximity to theinterface.

This surface selective property of SHG and other nonlinear opticaltechniques can be exploited to detect conformational change inbiological molecules immobilized at interfaces. For example,conformational change in a receptor molecule due to binding of a ligand,might be detected using a nonlinear-active label or moiety wherein thelabel is attached to or associated with the receptor such that theconformational change leads to a change in the orientation or distanceof the label with respect to the interface (FIG. 2), and thus to achange in a physical property of the nonlinear optical signal. Untilrecently, the use of surface-selective nonlinear optical techniques hasbeen confined mainly to applications in physics and chemistry, sincerelatively few biological samples are intrinsically non-linearly active.Recently, the use of second harmonic active labels (“SHG labels”) hasbeen introduced, allowing virtually any molecule or particle to berendered highly non-linear active. The first example of this wasdemonstrated by labeling the protein cytochrome c with an oxazole dyeand detecting the protein conjugate at an air-water interface withsecond harmonic generation [Salafsky, J., “‘SHG-labels’ for Detection ofMolecules by Second Harmonic Generation”, Chem. Phys. Lett.342(5-6):485-491 (2001)]. Examples of SHG data that demonstrate thedetection of spermine- or spermidine-induced conformational changes inalpha-synuclein are shown in FIGS. 3A-3D.

Surface-selective nonlinear optical techniques are also coherenttechniques, meaning that the fundamental and nonlinear optical lightbeams have wave fronts that propagate through space with well-definedspatial and phase relationships. The use of surface-selective nonlinearoptical detection techniques for analysis of conformation of biologicalmolecules or other biological entities has a number of inherentadvantages over other optical approaches, including: i) sensitive anddirect dependence of the nonlinear signal on the orientation and/ordipole moment(s) of the nonlinear-active species, thereby conferringsensitivity to conformational change; (ii) higher signal-to-noise (lowerbackground) than fluorescence-based detection since the nonlinearoptical signal is generated only at surfaces that create anon-centrosymmetric system, i.e. the technique inherently has a verynarrow “depth-of-field”; (iii) as a result of the narrow “depth offield”, the technique is useful when measurements must be performed inthe presence of a overlaying solution, e.g. where a binding processmight be obviated or disturbed by a separation or rinse step. Thisaspect of the technique may be particularly useful for performingequilibrium binding measurements, which require the presence of bulkspecies, or kinetics measurements where the measurements are made over adefined period of time; (iv) the technique exhibits lowerphoto-bleaching and heating effects than those that occur influorescence, due to the facts that the two-photon absorptioncross-section is typically much lower than the one-photon absorptioncross-section for a given molecule, and that SHG (and sum frequencygeneration or difference frequency generation) involves scattering, notabsorption; (v) minimal collection optics are required and higher signalto noise is expected since the fundamental and nonlinear optical beams(e.g., second harmonic light) have well-defined incoming and outgoingdirections with respect to the interface. This is particularlyadvantageous compared to fluorescence-based detection, as fluorescenceemission is isotropic and there may also be a large fluorescencebackground component to detected signals arising from out-of-focal planefluorescent species.

High Throughput Systems and Methods

Systems and methods are disclosed herein for implementing highthroughput analysis of conformation in biological entities based on theuse of second harmonic generation or related nonlinear optical detectiontechniques. As used herein, “high throughput” is a relative term used incomparison to structural measurements performed using traditionaltechniques such as NMR or X-ray crystallography. As will be described inmore detail below, the SHG-based methods and systems disclosed hereinare capable of performing structural determinations at a rate that is atleast an order-of-magnitude faster than these conventional techniques.

In one aspect, this disclosure provides a method for high throughputdetection of conformation or conformational change in one or morebiological entities, the method comprising (i) labeling one or moretarget biological entities, e.g. protein molecules, with anonlinear-active label or tag, (ii) immobilizing the one or more labeledtarget biological entities at one or more discrete regions of a planarsubstrate surface, wherein the substrate surface further comprises anoptical interface, (iii) sequentially exposing each discrete region toexcitation light by changing the position of the substrate relative toan external light source, (iv) collecting a nonlinear optical signalemitted from each discrete region as it is exposed to excitation light,and (v) processing said nonlinear optical signal to determine anorientation, conformation, or conformational change of each of the oneor more biological entities. In another aspect, the method furthercomprises (vi) contacting each of the one or more biological entitieswith one or more test entities following the first exposure toexcitation light, (vii) subsequently re-exposing each discrete region toexcitation light one or more times, (viii) collecting a nonlinearoptical signal from each discrete region as it is exposed to excitationlight, and (ix) processing said nonlinear optical signals to determinewhether or not a change in orientation or conformation has occurred inthe one or more biological entities as a result of contacting with saidone or more test entities. In one aspect of the method, nonlinearoptical signals are detected only once following contact of the one ormore biological entities with one or more test entities (i.e. endpointassay mode), and then used to determine whether or not conformationalchange has occurred. In another aspect, nonlinear optical signals arecollected repeatedly and at defined time intervals following contact ofthe one of more biological entities with one or more test entities (i.e.kinetics mode), and then used to determine the kinetics ofconformational change in the one or more biological entities. In apreferred aspect of the method, each discrete region of the substratecomprises a supported lipid bilayer structure, and biological entitiesare immobilized in each discrete region by means of tethering to orembedding in the lipid bilayer. In another preferred aspect of themethod, the excitation light is delivered to the substrate surface, i.e.the optical interface, by means of total internal reflection, and thenonlinear optical signals emitted from the discrete regions of thesubstrate surface are collected along the same optical axis as thereflected excitation light.

In order to implement high throughput analysis of conformation orconformational change using nonlinear optical detection, the systemsdescribed herein require several components (illustrated schematicallyin FIG. 4), including (i) at least one suitable excitation light sourceand optics for delivering the at least one excitation light beam to anoptical interface, (ii) an interchangeable substrate comprising theoptical interface, to which one or more biological entities have beentethered or immobilized in discrete regions of the substrate, (iii) ahigh-precision translation stage for positioning the substrate relativeto the at least one excitation light source, and (iv) optics forcollecting nonlinear optical signals generated as a result ofilluminating each of the discrete regions of the substrate withexcitation light and delivering said nonlinear signals to a detector,and (v) a processor for analyzing the nonlinear optical signal datareceived from the detector and determining conformation orconformational change for the one or more biological entitiesimmobilized on the substrate. In some aspects, the systems and methodsdisclosed herein further comprise the use of (vi) a programmablefluid-dispensing system for delivering test entities to each of thediscrete regions of the substrate, and (vii) the use of plate-handlingrobotics for automated positioning and replacement of substrates at theinterface with the optical system.

The methods and systems disclosed herein may be configured for analysisof a single biological entity contacted with a plurality of testentities, or for analysis of a plurality of biological entitiescontacted with a single test entity, or any combination thereof. Whencontacting one or more biological entities with a plurality of testentities, the contacting step may be performed sequentially, i.e. byexposing the immobilized biological entity to a single test entity for aspecified period of time, followed by an optional rinse step to removethe test entity solution and regenerate the immobilized biologicalentity prior to introducing to the next test entity, or the contactingstep may be performed in parallel, i.e. by having a plurality ofdiscrete regions comprising the same immobilized biological entity, andexposing the biological entity in each of the plurality of discreteregions to a different test entity. The methods and systems disclosedherein may be configured to perform analysis of conformational change inat least one biological entity, at least two biological entities, atleast four biological entities, at least six biological entities, atleast eight biological entities, at least ten biological entities, atleast fifteen biological entities, or at least twenty biologicalentities. In some aspects, methods and systems disclosed herein may beconfigured to perform analysis of conformational change in at mosttwenty biological entities, at most fifteen biological entities, at mostten biological entities, at most eight biological entities, at most sixbiological entities, at most four biological entities, at most twobiological entities, or at most one biological entity. Similarly, themethods and systems disclosed herein may be configured to performanalysis of conformational change upon exposure of the one or morebiological entities to at least 1 test entity, at least 5 test entities,at least 10 test entities, at least 50 test entities, at least 100 testentities, at least 500 test entities, at least 1,000 test entities, atleast 5,000 test entities, at least 10,000 test entities, or at least100,000 test entities. In some aspects, the methods and systemsdisclosed herein may be configured to perform analysis of conformationalchange upon exposure of the one or more biological test entities to atmost 100,000 test entities, at most 10,000 test entities, at most 5,000test entities, at most 1,000 test entities, at most 500 test entities,at most 100 test entities, at most 50 test entities, at most 10 testentities, at most 5 test entities, or at most 1 test entity.

Biological Entities and Test Entities

As used herein, the phrase “biological entities” comprises but is notlimited to cells, proteins, peptides, receptors, enzymes, antibodies,DNA, RNA, biological molecules, oligonucleotides, solvents, smallmolecules, synthetic molecules, carbohydrates, or any combinationthereof. Similarly, the phrase “test entities” also comprises but is notlimited to cells, proteins, peptides, receptors, enzymes, antibodies,DNA, RNA, biological molecules, oligonucleotides, solvents, smallmolecules, synthetic molecules, carbohydrates, or any combinationthereof. In some aspects, biological entities may comprise drug targets,or portions thereof, while test entities may comprise drug candidates,or portions thereof.

Nonlinear-Active Labels and Labeling Techniques

As noted above, most biological molecules are not intrinsicallynonlinear-active. Exceptions include collagen, a structural protein thatis found in most structural or load-bearing tissues. SHG microscopy hasbeen used extensively in studies of collagen-containing structures, forexample, the cornea. Other biological molecules or entities must berendered nonlinear-active by means of introducing a nonlinear-activemoiety such as a tag or label. A label for use in the present inventionrefers to a nonlinear-active moiety, tag, molecule, or particle whichcan be bound, either covalently or non-covalently to a molecule,particle or phase (e.g., a lipid bilayer) in order to render theresulting system more nonlinear optical active. Labels can be employedin the case where the molecule, particle or phase (e.g., lipid bilayer)is not nonlinear active to render the system nonlinear-active, or with asystem that is already nonlinear-active to add an extra characterizationparameter into the system. Exogenous labels can be pre-attached to themolecules, particles, or other biological entities, and any unbound orunreacted labels separated from the labeled entities before use in themethods described herein. In a specific aspect of the methods disclosedherein, the nonlinear-active moiety is attached to the target moleculeor biological entity in vitro prior to immobilizing the target moleculesor biological entities in discrete regions of the substrate surface. Thelabeling of biological molecules or other biological entities withnonlinear-active labels allows a direct optical means of detectinginteractions between the labeled biological molecule or entity andanother molecule or entity (i.e. the test entity) in cases where theinteraction results in a change in orientation or conformation of thebiological molecule or entity using a surface-selective nonlinearoptical technique.

In alternative aspects of the methods and systems described herein, atleast two distinguishable nonlinear-active labels are used. Theorientation of the attached two or more distinguishable labels wouldthen be chosen to facilitate well defined directions of the emanatingcoherent nonlinear light beam. The two or more distinguishable labelscan be used in assays where multiple fundamental light beams at one ormore frequencies, incident with one or more polarization directionsrelative to the optical interface are used, with the resulting emanationof at least two nonlinear light beams.

Examples of nonlinear-active tags or labels include, but are not limitedto, the compounds listed in Table 1, and their derivatives.

TABLE 1 Examples of Nonlinear-Active Tags 2-aryl-5-(4- HemicyaninesPolyimides pyridyl)oxazole 2-(4-pyridyl)- lndandione-1,3-Polymethacrylates cyclo- pyidinium betaine alkano[d]oxazoles5-aryl-2-(4- lndodicarbocyanines PyMPO pyridyl)oxazole (pyridyloxazole)7-Hydroxycoumarin-3- Melamines PyMPO, succinimidyl carboxylic acid,ester (1-(3- succinimidyl (succinimidyloxy- ester carbonyl)benzyl)-4-(5-(4-PyMPO, maleimide Azo dyes Merocyanines Stilbazims BenzooxazolesMethoxyphenyl)oxazol- Stilbenes 2-yl)pyridinium bromide) BithiophenesMethylene blue Stryryl-based dyes Cyanines Oxazole or oxadizoleSulphonyl-substituted molecules azobenzenes Dapoxyl Oxonols Thiophenescarboxylic acid, succinimidyl ester Diaminobenzene PerylenesTricyanovinyl aniline compounds Diazostilbenes Phenothiazine-stilbazoleTricyanovinyl azo Fluoresceins Polyenes

In evaluating whether a species may be nonlinear-active, the followingcharacteristics can indicate the potential for nonlinear activity: alarge difference dipole moment (difference in dipole moment between theground and excited states of the molecule), a large Stokes shift influorescence, or an aromatic or conjugated bonding character. In furtherevaluating such a species, an experimenter can use a simple techniqueknown to those skilled in the art to confirm the nonlinear activity, forexample, through detection of SHG from an air-water interface on whichthe nonlinear-active species has been distributed. Once a suitablenonlinear-active species bas been selected for the experiment at hand,the species can be conjugated, if desired, to a biological molecule orentity for use in the surface-selective nonlinear optical methods andsystems disclosed herein.

The following reference and references therein describe techniquesavailable for creating a labeled biological entity from a synthetic dyeand many other molecules: Greg T. Hermanson, Bioconjugate Techniques,Academic Press, New York, 1996.

In a specific aspect of the methods and systems disclosed, metalnanoparticles and assemblies thereof are modified to create biologicalnonlinear-active labels. The following references describe themodification of metal nanoparticles and assemblies: J. P. Novak and D.L. Feldheim, “Assembly of Phenylacetylene-Bridged Silver and GoldNanoparticle Arrays”, J. Am. Chem. Soc. 122:3979-3980 (2000); J. P.Novak, et al., “Nonlinear Optical Properties of Molecularly Bridged GoldNanoparticle Arrays”, J. Am. Chem. Soc. 122:12029-12030 (2000); Vance,F. W., Lemon, B. I., and Hupp, J. T., “Enormous Hyper-RayleighScattering from Nanocrystalline Gold Particle Suspensions”, J. Phys.Chem. B 102:10091-93 (1999).

In yet another aspect of the methods and systems disclosed herein, thenonlinear activity of the system can also be manipulated through theintroduction of nonlinear analogues to molecular beacons, that is,molecular beacon probes that have been modified to incorporate anonlinear-active label (or modulator thereof) instead of fluorophoresand quenchers. These nonlinear optical analogues of molecular beaconsare referred to herein as molecular beacon analogues (MB analogues orMBA). The MB analogues to be used in the described methods and systemscan be synthesized according to procedures known to one of ordinaryskill in the art.

Types of Biological Interactions Detected

The methods and systems disclosed herein provide for detection of avariety of interactions between biological entities, or betweenbiological entities and test entities, depending on the choice ofbiological entities, test entities, and non-linear active labelingtechnique employed. In one aspect, the present disclosure provides forthe qualitative detection of binding events, e.g. the binding of aligand to a receptor, as indicated by the resulting conformationalchange induced in the receptor. In another aspect, the presentdisclosure provides for quantitative analysis of binding events, e.g.the binding of a ligand to a receptor, by performing replicatemeasurements using different concentrations of the ligand molecule andgenerating a dose-response curve using the percent change in maximalconformational change observed. Similarly, other aspects of the presentdisclosure may provide methods for qualitative or quantitativemeasurements of enzyme-inhibitor interactions, antibody-antigeninteractions, the formation of complexes of biological macromolecules,or interactions of receptors with allosteric modulators.

In other specific embodiments, MB analogues can be used according to themethods disclosed herein as hybridization probes that can detect thepresence of complementary nucleic acid target without having to separateprobe-target hybrids from excess probes as in solution-phasehybridization assays, and without the need to label the targetsoligonucleotides. MB analogue probes can also be used for the detectionof RNAs within living cells, for monitoring the synthesis of specificnucleic acids in sample aliquots drawn from bioreactors, and for theconstruction of self-reporting oligonucleotide arrays. They can be usedto perform homogeneous one-well assays for the identification ofsingle-nucleotide variations in DNA and for the detection of pathogensor cells immobilized to surfaces for interfacial detection.

Interactions between biological entities or biological and test entities(e.g. binding reactions, conformational changes, etc.) can be correlatedthrough the methods presently disclosed to the following measurablenonlinear signal parameters: (i) the intensity of the nonlinear light,(ii) the wavelength or spectrum of the nonlinear light, (iii) thepolarization of the nonlinear light, (iv) the time-course of (i), (ii),or (iii), and/or vi) one or more combinations of (i), (ii), (iii), and(iv).

Laser Light Sources and Excitation Optical System

FIG. 5 illustrates one aspect of the methods and systems disclosedherein wherein second harmonic light is generated by reflecting incidentfundamental excitation light from the surface of a substrate comprisingthe sample interface (or optical interface). FIG. 6 shows a photographof one example of a suitable optical setup. A laser provides thefundamental light necessary to generate second harmonic light at thesample interface. Typically this will be a picosecond or femtosecondlaser, either wavelength tunable or not tunable, and commerciallyavailable (e.g. a Ti: Sapphire femtosecond laser or fiber laser system).Light at the fundamental frequency (w) exits the laser and itspolarization is selected using, for example a half-wave plateappropriate to the frequency and intensity of the light (e.g., availablefrom Melles Griot, Oriel, or Newport Corp.). The beam then passesthrough a harmonic separator designed to pass the fundamental light butblock nonlinear light (e.g. second harmonic light). This filter is usedto prevent back-reflection of the second harmonic beam into the lasercavity which can cause disturbances in the lasing properties. Acombination of mirrors and lenses are then used to steer and shape thebeam prior to reflection from a final mirror that directs the beam via aprism to impinge at a specific location and with a specific angle θ onthe substrate surface such that it undergoes total internal reflectionat the substrate surface. One of the mirrors in the optical path can bescanned if required using a galvanometer-controlled mirror scanner, arotating polygonal mirror scanner, a Bragg diffractor, acousto-opticdeflector, or other means known in the art to allow control of amirror's position. The substrate comprising the optical interface andnonlinear-active sample surface can be mounted on an x-y translationstage (computer controlled) to select a specific location on thesubstrate surface for generation of the second harmonic beam. In someaspects of the methods and systems presently described, it is desirableto scan or rotate one mirror in order to slightly vary the angle ofincidence for total internal reflection, and thereby maximize thenonlinear optical signal emitted from the discrete regions of thesubstrate surface without substantially changing the position of theilluminating excitation light spot. In some aspects, two (or more)lasers having different fundamental frequencies may be used to generatesum frequency or difference frequency light at the optical interface onwhich the non-linear active sample is immobilized.

Substrate Formats, Optical Interface, and Total Internal Reflection

As described above, the systems and methods of the present disclosureutilize a planar substrate for immobilization of one or more biologicalentities on a top surface of the substrate, wherein the top substratesurface further comprises the optical interface (or sample interface)used for exciting nonlinear optical signals. The substrate can be glass,silica, fused-silica, plastic, or any other solid material that istransparent to the fundamental and second harmonic light beams, and thatsupports total internal reflection at the substrate/sample interfacewhen the excitation light is incident at an appropriate angle. In someaspects of the invention, the discrete regions within which biologicalentities are contained are configured as one-dimensional ortwo-dimensional arrays, and are separated from one another by means of ahydrophobic coating or thin metal layer. In other aspects, the discreteregions may comprise indents in the substrate surface. In still otheraspects, the discrete regions may be separated from each other by meansof a well-forming component such that the substrate forms the bottom ofa microwell plate (or microplate), and each individual discrete regionforms the bottom of one well in the microwell plate. In one aspect ofthe present disclosure, the well-forming component separates the topsurface of the substrate into 96 separate wells. In another aspect, thewell-forming component separates the top surface of the substrate into384 wells. In yet another aspect, the well-forming component separatesthe top surface of the substrate into 1,536 wells. In all of theseaspects, the substrate, whether configured in a planar array, indentedarray, or microwell plate format, may comprise a disposable orconsumable device or cartridge that interfaces with other optical andmechanical components of the high throughput system.

The methods and systems disclosed herein further comprise specifying thenumber of discrete regions or wells into which the substrate surface isdivided, irrespective of how separation is maintained between discreteregions or wells. Having larger numbers of discrete regions or wells ona substrate may be advantageous in terms of increasing the sampleanalysis throughput of the method or system. In one aspect of thepresent disclosure, the number of discrete regions or wells persubstrate is between 10 and 1,600. In other aspects, the number ofdiscrete regions or wells is at least 10, at least 20, at least 50, atleast 100, at least 200, at least 300, at least 400, at least 500, atleast 750, at least 1,000, at least 1,250, at least 1,500, or at least1,600. In yet other aspects of the disclosed methods and systems, thenumber of discrete regions or wells is at most 1,600, at most 1,500, atmost 1,000, at most 750, at most 500, at most 400, at most 300, at most200, at most 100, at most 50, at most 20, or at most 10. In a preferredaspect, the number of discrete regions or wells is 96. In anotherpreferred aspect, the number of discrete regions or wells is 384. In yetanother preferred aspect, the number of discrete regions or wells is1,536. Those of skill in the art will appreciate that the number ofdiscrete regions or wells may fall within any range bounded by any ofthese values (e.g. from about 12 to about 1,400).

The methods and systems disclosed herein also comprise specifying thesurface area of the discrete regions or wells into which the substratesurface is divided, irrespective of how separation is maintained betweendiscrete regions or wells. Having discrete regions or wells of largerarea may facilitate ease-of-access and manipulation of the associatedbiological entities in some cases, whereas having discrete regions orwells of smaller area may be advantageous in terms of reducing assayreagent volume requirements and increasing the sample analysisthroughput of the method or system. In one aspect of the presentdisclosure, the surface area of the discrete regions or wells is between1 mm² and 100 mm². In other aspects, the area of the discrete regions orwells is at least 1 mm², at least 2.5 mm², at least 5 mm², at least 10mm², at least 20 mm², at least 30 mm², at least 40 mm², at least 50 mm²,at least 75 mm², or at least 100 mm². In yet other aspects of thedisclosed methods and systems, the area of the discrete regions or wellsis at most 100 mm², at most 75 mm², at most 50 mm², at most 40 mm², atmost 30 mm², at most 20 mm², at most 10 mm², at most 5 mm², at most 2.5mm², or at most 1 mm². In a preferred aspect, the area of discreteregions or wells is about 35 mm². In another preferred aspect, the areaof the discrete regions or wells is about 8.6 mm². Those of skill in theart will appreciate that the area of the discrete regions or wells mayfall within any range bounded by any of these values (e.g. from about 2mm² to about 95 mm²).

Discrete regions of the substrate surface are sequentially exposed to(illuminated with) excitation light by re-positioning the substraterelative to the excitation light source. Total internal reflection ofthe incident excitation light creates an “evanescent wave” at the sampleinterface, which excites the nonlinear-active label and results ingeneration of second harmonic light (or in some aspects, sum frequencyor difference frequency light). Because the intensity of the evanescentwave, and hence the intensity of the nonlinear optical signalsgenerated, is dependent on the incident angle of the excitation lightbeam, precise orientation of the substrate plane with respect to theoptical axis of the excitation beam and efficient optical coupling ofthe beam to the substrate is critical for achieving optimal SHG signalacross the array of discrete regions. In some aspects of the presentdisclosure, total internal reflection is achieved by means of a singlereflection of the excitation light from the substrate surface. In otheraspects, the substrate may be configured as a waveguide such that theexcitation light undergoes multiple total internal reflections as itpropagates along the waveguide. In yet other aspects, the substrate maybe configured as a zero-mode waveguide, wherein an evanescent field iscreated by means of nanofabricated structures.

Efficient optical coupling between the excitation light beam and thesubstrate in an optical setup such as the one illustrated in FIGS. 5 and7 would typically be achieved by use of an index-matching fluid such asmineral oil, mixtures of mineral oil and hydrogenated terphenyls,perfluorocarbon fluids, glycerin, glycerol, or similar fluids having arefractive index near 1.5, wherein the index-matching fluid is wickedbetween the prism and the lower surface of the substrate. Since astatic, bubble-free film of index-matching fluid is likely to bedisrupted during fast re-positioning of the substrate, the systems andmethods disclosed herein include alternative approaches for creatingefficient optical coupling of the excitation beam to the substrate inhigh throughput systems.

FIG. 8 illustrates one aspect of a high throughput system of the presentdisclosure in which a continuously recirculating stream ofindex-matching fluid is used to maintain efficient optical couplingbetween the prism, which is mounted as part of the optical system, andthe substrate, which is configured in a microwell plate format (e.g. aglass bottom microplate format) and is free to translate relative to theprism. The continuous flow of index-matching fluid in this case ensuresthat the thin film of fluid between the prism and substrate is neverdisrupted as the two components move relative to each other, i.e. anysmall bubbles or discontinuities in the thin layer of fluid will beeliminated or pushed out from the gap between the prism and substrate bymeans of the fluid flow. Index-matching fluid is introduced into the gapvia the two fluid channels indicated in the drawing, and may becollected in a suitable reservoir or sump, from which it may berecirculated using a small pump. In an alternative implementation of thesame concept, instead of the line-contact between substrate and prismindicated in FIG. 8, point contact between a single discrete region anda cylindrical total internal reflection (TIR) probe would be utilized,where the index-matching fluid would flow up through a center fluidchannel, and then down over the sides of the cylindrical TIR probe to becollected in a suitable reservoir or sump.

FIG. 9 illustrates another aspect of a high throughput system of thepresent disclosure, in which a thin layer of index-matching elastomericmaterial is used in place of index-matching fluid to maintain efficientoptical coupling between the prism and substrate. In this case, thesubstrate is again packaged in a microwell plate format (e.g. a glassbottom microplate format), but with a thin layer of an index-matchingelastomeric material attached to or adjacent to the lower surface of thesubstrate, such that when placed in contact with the upper surface ofthe prism, the elastomer fills the gap between prism and substrate andprovides for efficient optical coupling. Examples of elastomericmaterials that may be used include, but are not limited to siliconeshaving a refractive index of about 1.4. In one aspect of the presentdisclosure, the refractive index of the elastomeric material is betweenabout 1.35 and about 1.6. In other aspects, the index of refraction isabout 1.6 or less, about 1.55 or less, about 1.5 or less, about 1.45 orless, about 1.4 or less, or about 1.35 or less. In yet other aspects,the index of refraction is at least about 1.35, at least about 1.4, atleast about 1.45, at least about 1.5, at least about 1.55, or at leastabout 1.6. Those of skill in the art will appreciate that the index ofrefraction of the elastomeric layer may fall within any range bounded byany of these values (e.g. from about 1.4 to about 1.6). In one aspect ofthis approach, the thickness of the layer of elastomeric material isbetween about 0.1 mm and 2 mm. In other aspects, the thickness of theelastomeric layer is at least 0.1 mm, at least 0.2 mm, at least 0.4 mm,at least 0.6 mm, at least 0.8 mm, at least 1.0 mm, at least 1.2 mm, atleast 1.4 mm, at least 1.6 mm, at least 1.8 mm, or at least 2.0 mm. Inanother aspect of this approach, the thickness of the elastomeric layeris at most 2.0 mm, at most 1.8 mm, at most 1.6 mm, at most 1.4 mm, atmost 1.2 mm, at most 1.0 mm, at most 0.8 mm, at most 0.6 mm, at most 0.4mm, at most 0.2 mm, or at most 0.1 mm. Those of skill in the art willappreciate that the thickness of the elastomeric layer my fall withinany range bounded by any of these values (e.g. from about 0.1 mm toabout 1.5 mm). In another aspect of this approach, the upper surface ofthe prism has a partially-cylindrical ridge or is domed (FIG. 9) tofocus the compression force and provide better contact betweensubstrate, elastomeric layer, and prism surface. This approach may alsorequire the use of a third axis of translation for positioning of thesubstrate, i.e. between excitation and detection steps, the substrate(microwell plate) would be raised slightly to eliminate contact betweenthe elastomeric layer and the prism prior to re-positioning thesubstrate to the location of the next discrete region to be analyzed.

FIGS. 10A-D illustrate a preferred aspect of a high throughput system ofthe present disclosure, in which an array of prisms or gratings isintegrated with the lower surface of the substrate (packaged in amicrowell plate format) and used to replace the fixed prism, therebyeliminating the need for index-matching fluids or elastomeric layersentirely. The array of prisms (or gratings) is aligned with the array ofdiscrete regions or wells on the upper surface of the substrate in sucha way that incident excitation light is directed by an “entrance prism”(“entrance grating”) to a discrete region or well that is adjacent tobut not directly above the entrance prism (entrance grating), at anangle of incidence that enables total internal reflection of theexcitation light beam from the sample interface (see FIG. 11), and suchthat the reflected excitation beam, and nonlinear-optical signalsgenerated at the illuminated discrete region, are collected by an “exitprism” (“exit grating”) that is again offset from (adjacent to but notdirectly underneath) the discrete region under interrogation, andwherein the entrance prism and exit prism (entrance grating and exitgrating) for each discrete region are different, non-unique elements ofthe array.

In general, for an array of discrete regions comprising M rows×N columnsof individual features, the corresponding prism or grating array willhave M+2 rows×N columns or N+2 columns×M rows of individual prisms orgratings. In some embodiments, M may have a value of at least 2, atleast 4, at least 6, at least 8, at least 12, at least 14, at least 16,at least 18, at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, or at least 50 rows. In some embodiments, M mayhave a value of at most 50, at most 45, at most 40, at most 35, at most30, at most 25, at most 20, at most 18, at most 16, at most 14, at most12, at most 10, at most 8, at most 6, at most 4, or at most 2 rows.Similarly, in some embodiments, N may have a value of at least 2, atleast 4, at least 6, at least 8, at least 12, at least 14, at least 16,at least 18, at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, or at least 50 columns. In some embodiments, Nmay have a value of at most 50, at most 45, at most 40, at most 35, atmost 30, at most 25, at most 20, at most 18, at most 16, at most 14, atmost 12, at most 10, at most 8, at most 6, at most 4, or at most 2columns. As will be apparent to those of skill in the art, M and N mayhave the same value or different values, and may have any value withinthe range specified above, for example, M=15 and N=45.

The geometry and dimensions of the individual prisms or gratings,including the thickness of the prism or grating array layer, areoptimized to ensure that incident light undergoes total internalreflection at the selected discrete region of the substrate, andnonlinear optical signals generated at the selected discrete region arecollected, with high optical coupling efficiency, independently of theposition of substrate (microwell plate) relative to the excitation lightbeam. FIG. 12 shows a photograph of a prism array prototype. The prismor grating arrays may be fabricated by a variety of techniques known tothose of skill in the art, for example, in a preferred aspect, they maybe injection molded from smooth flowing, low birefringence materialssuch as cyclic olefin copolymer (COC) or cyclic olefin polymer (COP),acrylic, polyester, or similar polymers. In some aspects, the prism orgrating array may be fabricated as a separate component, andsubsequently integrated with the lower surface of the substrate. Inother aspects, the prism or grating array may be fabricated as anintegral feature of substrate itself.

Immobilization Chemistries

As disclosed herein, substrates in any of the formats described aboveare further configured for immobilization of biological entities withinthe specified discrete regions. Immobilization of biological moleculesor cells may be accomplished by a variety of techniques known to thoseof skill in the art, for example, through the use of aminopropyl silanechemistries to functionalize glass or fused-silica surfaces with aminefunctional groups, followed by covalent coupling using amine-reactiveconjugation chemistries, either directly with the biological molecule ofinterest, or via an intermediate spacer or linker molecule. Non-specificadsorption may also be used directly or indirectly, e.g. through the useof BSA-NHS (BSA-N-hydroxysuccinimide) by first attaching a molecularlayer of BSA to the surface and then activating it withN,N′-disuccinimidyl carbonate. The activated lysine, aspartate orglutamate residues on the BSA react with surface amines on proteins.

In a preferred aspect of the present disclosure, biological moleculesmay be immobilized on the surface by means of tethering to or embeddingin “supported lipid bilayers”, the latter comprising small patches oflipid bilayer confined to a silicon or glass surface by means ofhydrophobic and electrostatic interactions, where the bilayer is“floating” above the substrate surface on a thin layer of aqueousbuffer. Supported phospholipid bilayers can also be prepared with orwithout membrane proteins or other membrane-associated components asdescribed, for example, in Salafsky et al., “Architecture and Functionof Membrane Proteins in Planar Supported Bilayers: A Study withPhotosynthetic Reaction Centers”, Biochemistry 35 (47): 14773-14781(1996); Gennis, R., Biomembranes, Springer-Verlag, 1989; Kalb et al.,“Formation of Supported Planar Bilayers by Fusion of Vesicles toSupported Phospholipid Monolayers”, Biochimica Biophysica Acta.1103:307-316 (1992); and Brian et al. “Allogeneic Stimulation ofCytotoxic T-cells by Supported Planar Membranes”, PNAS-BiologicalSciences 81(19): 6159-6163 (1984), relevant portions of which areincorporated herein by reference. Supported phospholipid bilayers arewell known in the art and there are numerous techniques available fortheir fabrication. Supported bilayers should typically be submerged inaqueous solution to prevent their destruction when exposed to air.

Collection Optics and Detector

FIG. 5 further illustrates the collection optics and detector used todetect nonlinear optical signals generated upon sequential illuminationof the discrete regions of the substrate. Because surface-selectivenonlinear optical techniques are coherent techniques, meaning that thefundamental and nonlinear optical light beams have wave fronts thatpropagate through space with well-defined spatial and phaserelationships, minimal collection optics are required. Emitted nonlinearoptical signals are collected by means of a prism (or the integratedprism or grating array of the microplate device described above) anddirected via a dichroic reflector and mirror to the detector. Additionaloptical components, e.g. lenses, optical bandpass filters, mirrors, etc.are optionally used to further shape, steer, and/or filter the beamprior to reaching the detector. A variety of different photodetectorsmay be used, including but not limited to photodiodes, avalanchephotodiodes, photomultipliers, CMOS sensors, or CCD devices.

X-Y Translation Stage

As illustrated in FIG. 4, implementation of the high throughput systemsdisclosed herein ideally utilizes a high precision X-Y (or in somecases, an X-Y-Z) translation stage for re-positioning the substrate (inany of the formats described above) in relation to the excitation lightbeam. Suitable translation stages are commercially available from anumber of vendors, for example, Parker Hannifin. Precision translationstage systems typically comprise a combination of several componentsincluding, but not limited to, linear actuators, optical encoders, servoand/or stepper motors, and motor controllers or drive units. Highprecision and repeatability of stage movement is required for thesystems and methods disclosed herein in order to ensure accuratemeasurements of nonlinear optical signals when interspersing repeatedsteps of optical detection and/or liquid-dispensing. Also, as the sizeof the focal spot for the excitation light [20-200 microns in diameteror on a side is substantially smaller than the size of the discreteregions on the substrate, in some aspects of the present disclosure, itmay also be desirable to return to a slightly different position withina given discrete region when making replicate measurements, or to slowlyscan the excitation beam across a portion of the discrete region overthe course of a single measurement, thereby eliminating potentialconcerns regarding the photo-bleaching effects of long exposures orprior exposures.

Consequently, the methods and systems disclosed herein further comprisespecifying the precision with which the translation stage is capable ofpositioning a substrate in relation to the excitation light beam. In oneaspect of the present disclosure, the precision of the translation stageis between about 1 um and about 10 um. In other aspects, the precisionof the translation stage is about 10 um or less, about 9 um or less,about 8 um or less, about 7 um or less, about 6 um or less, about 5 umor less, about 4 um or less, about 3 um or less, about 2 um or less, orabout 1 um or less. Those of skill in the art will appreciate that theprecision of the translation stage may fall within any range bounded byany of these values (e.g. from about 1.5 um to about 7.5 um).

Fluid Dispensing System

As illustrated in FIG. 4, some embodiments of the high throughputsystems disclosed herein further comprise an automated, programmablefluid-dispensing (or liquid-dispensing) system for use in contacting thebiological or target entities immobilized on the substrate surface withtest entities (or test compounds), the latter typically being dispensedin solutions comprising aqueous buffers with or without the addition ofa small organic solvent component, e.g. dimethylsulfoxide (DMSO).Suitable automated, programmable fluid-dispensing systems arecommercially available from a number of vendors, e.g. Beckman Coulter,Perkin Elmer, Tecan, Velocity 11, and many others. In a preferred aspectof the systems and methods disclosed herein, the fluid-dispensing systemfurther comprises a multichannel dispense head, e.g. a 4 channel, 8channel, 16 channel, 96 channel, or 384 channel dispense head, forsimultaneous delivery of programmable volumes of liquid (e.g. rangingfrom about 1 microliter to several milliliters) to multiple wells orlocations on the substrate.

Plate-Handling Robotics

In other aspects of the high throughput systems disclosed herein, thesystem further comprises a microplate-handling (or plate-handling)robotic system (FIG. 4) for automated replacement and positioning ofsubstrates (in any of the formats described above) in relation to theoptical excitation and detection optics, or for optionally movingsubstrates between the optical instrument and the fluid-dispensingsystem. Suitable automated, programmable microplate-handling roboticsystems are commercially available from a number of vendors, includingBeckman Coulter, Perkin Elemer, Tecan, Velocity 11, and many others. Ina preferred aspect of the systems and methods disclosed herein, theautomated microplate-handling robotic system is configured to movecollections of microwell plates comprising immobilized biologicalentities and/or aliquots of test compounds to and from refrigeratedstorage units.

Processor/Controller and Constraint-Based Scheduling Algorithm

In another aspect of the present disclosure, the high throughput systemsdisclosed further comprise a processor (or controller, or computersystem) (FIG. 4) configured to run system software which controls thevarious subsystems described (excitation and detection optical systems,X-Y (or X-Y-Z) translation stage, fluid-dispensing system, andplate-handling robotics) and synchronizes the different operationalsteps involved in performing high throughput conformational analysis. Inaddition to handling the data acquisition process, i.e. collection ofoutput electronic signals from the detector that correspond to thenonlinear optical signals associated with conformational change, theprocessor or controller is also typically configured to store the data,perform data processing and display functions (including determinationof whether or not changes in orientation or conformation have occurredfor the biological entities, or combinations of biological and testentities, that have been tested), and operate a graphical user interfacefor interactive control by an operator. The processor or controller mayalso be networked with other processors, or connected to the internetfor communication with other instruments and computers at remotelocations.

Typical input parameters for the processor/controller may include set-upparameters such as the total number of microwell plates to be analyzed;the number of wells per plate; the number of times excitation anddetection steps are to be performed for each discrete region of thesubstrate or well of the microplate (e.g. to specify endpoint assay orkinetic assay modes); the total timecourse over which kinetic datashould be collected for each discrete region or well; the order, timing,and volume of test compound solutions to be delivered to each discreteregion or well; the dwell time for collection and integration ofnonlinear optical signals; the name(s) of output data files; and any ofa number of system set-up and control parameters known to those skilledin the art.

In a preferred aspect of the present disclosure, the processor orcontroller is further configured to perform system throughputoptimization by means of executing a constraint-based schedulingalgorithm. This algorithm utilizes system set-up parameters as describedabove to determine an optimal sequence of interspersedexcitation/detection and liquid-dispensing steps for discrete regions orwells that may or may not be adjacent to each other, such that theoverall throughput of the system, in terms of number of biologicalentities and/or test entities analyzed per hour, is maximized.Optimization of system operational steps is an important aspect ofachieving high throughput analysis. In some aspects of the disclosedmethods and systems, the average throughput of the analysis system mayrange from about 10 test entities tested per hour to about 1,000 testentities tested per hour. In some aspects, the average throughput of theanalysis system may be at least 10 test entities tested per hour, atleast 25 test entities tested per hour, at least 50 test entities testedper hour, at least 75 test entities tested per hour, at least 100 testentities tested per hour, at least 200 test entities tested per hour, atleast 400 test entities tested per hour, at least 600 test entitiestested per hour, at least 800 test entities tested per hour, or at least1,000 test entities tested per hour. In other aspects, the averagethroughput of the analysis system may be at most 1,000 test entitiestested per hour, at most 800 test entities tested per hour, at most 600test entities tested per hour, at most 400 test entities tested perhour, at most 200 test entities tested per hour, at most 100 testentities tested per hour, at most 75 test entities tested per hour, atmost 50 test entities tested per hour, at most 25 test entities testedper hour, or at most 10 test entities tested per hour.

Computer Systems and Networks

In various embodiments, the methods and systems of the invention mayfurther comprise software programs installed on computer systems and usethereof. Accordingly, as noted above, computerized control of thevarious subsystems and synchronization of the different operationalsteps involved in performing high throughput conformational analysis,including data analysis and display, are within the bounds of theinvention.

The computer system 500 illustrated in FIG. 21 may be understood as alogical apparatus that can read instructions from media 511 and/or anetwork port 505, which can optionally be connected to server 509 havingfixed media 512. The system, such as shown in FIG. 21 can include a CPU501, disk drives 503, optional input devices such as keyboard 515 and/ormouse 516 and optional monitor 507. Data communication can be achievedthrough the indicated communication medium to a server at a local or aremote location. The communication medium can include any means oftransmitting and/or receiving data. For example, the communicationmedium can be a network connection, a wireless connection or an internetconnection. Such a connection can provide for communication over theWorld Wide Web. It is envisioned that data relating to the presentdisclosure can be transmitted over such networks or connections forreception and/or review by a party 522 as illustrated in FIG. 21.

FIG. 22 is a block diagram illustrating a first example architecture ofa computer system 100 that can be used in connection with exampleembodiments of the present invention. As depicted in FIG. 22, theexample computer system can include a processor 102 for processinginstructions. Non-limiting examples of processors include: the IntelXeon™ processor, the AMD Opteron™ processor, the Samsung 32-bit RISC ARM1176JZ(F)-S v1.0™ processor, the ARM Cortex-A8 Samsung S5PC100™processor, the ARM Cortex-A8 Apple A4™ processor, the Marvell PXA 930™processor, or a functionally-equivalent processor. Multiple threads ofexecution can be used for parallel processing. In some embodiments,multiple processors or processors with multiple cores can also be used,whether in a single computer system, in a cluster, or distributed acrosssystems over a network comprising a plurality of computers, cell phones,and/or personal data assistant devices.

As illustrated in FIG. 22, a high speed cache 104 can be connected to,or incorporated in, the processor 102 to provide a high speed memory forinstructions or data that have been recently, or are frequently, used byprocessor 102. The processor 102 is connected to a north bridge 106 by aprocessor bus 108. The north bridge 106 is connected to random accessmemory (RAM) 110 by a memory bus 112 and manages access to the RAM 110by the processor 102. The north bridge 106 is also connected to a southbridge 114 by a chipset bus 116. The south bridge 114 is, in turn,connected to a peripheral bus 118. The peripheral bus can be, forexample, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and south bridge are often referred to as a processor chipset andmanage data transfer between the processor, RAM, and peripheralcomponents on the peripheral bus 118. In some alternative architectures,the functionality of the north bridge can be incorporated into theprocessor instead of using a separate north bridge chip.

In some embodiments, system 100 can include an accelerator card 122attached to the peripheral bus 118. The accelerator can include fieldprogrammable gate arrays (FPGAs) or other hardware for acceleratingcertain processing. For example, an accelerator can be used for adaptivedata restructuring or to evaluate algebraic expressions used in extendedset processing.

Software and data are stored in external storage 124 and can be loadedinto RAM 110 and/or cache 104 for use by the processor. The system 100includes an operating system for managing system resources; non-limitingexamples of operating systems include: Linux, Windows, MacOS™,BlackBerry OS™, iOS™, and other functionally-equivalent operatingsystems, as well as application software running on top of the operatingsystem for managing data storage and optimization in accordance withexample embodiments of the present invention.

In this example, system 100 also includes network interface cards (NICs)120 and 121 connected to the peripheral bus for providing networkinterfaces to external storage, such as Network Attached Storage (NAS)and other computer systems that can be used for distributed parallelprocessing.

FIG. 23 is a diagram showing a network 200 with a plurality of computersystems 202 a, and 202 b, a plurality of cell phones and personal dataassistants 202 c, and Network Attached Storage (NAS) 204 a, and 204 b.In example embodiments, systems 202 a, 202 b, and 202 c can manage datastorage and optimize data access for data stored in Network AttachedStorage (NAS) 204 a and 204 b. A mathematical model can be used for thedata and be evaluated using distributed parallel processing acrosscomputer systems 202 a, and 202 b, and cell phone and personal dataassistant systems 202 c. Computer systems 202 a, and 202 b, and cellphone and personal data assistant systems 202 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 204 a and 204 b. FIG. 23 illustratesan example only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various embodiments of thepresent invention. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface.

In some example embodiments, processors can maintain separate memoryspaces and transmit data through network interfaces, back plane or otherconnectors for parallel processing by other processors. In otherembodiments, some or all of the processors can use a shared virtualaddress memory space.

FIG. 24 is a block diagram of a multiprocessor computer system 300 usinga shared virtual address memory space in accordance with an exampleembodiment. The system includes a plurality of processors 302 a-f thatcan access a shared memory subsystem 304. The system incorporates aplurality of programmable hardware memory algorithm processors (MAPs)306 a-f in the memory subsystem 304. Each MAP 306 a-f can comprise amemory 308 a-f and one or more field programmable gate arrays (FPGAs)310 a-f. The MAP provides a configurable functional unit and particularalgorithms or portions of algorithms can be provided to the FPGAs 310a-f for processing in close coordination with a respective processor.For example, the MAPs can be used to evaluate algebraic expressionsregarding the data model and to perform adaptive data restructuring inexample embodiments. In this example, each MAP is globally accessible byall of the processors for these purposes. In one configuration, each MAPcan use Direct Memory Access (DMA) to access an associated memory 308a-f, allowing it to execute tasks independently of, and asynchronouslyfrom, the respective microprocessor 302 a-f. In this configuration, aMAP can feed results directly to another MAP for pipelining and parallelexecution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleembodiments, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some embodiments,all or part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example embodiments, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example embodiments, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other embodiments, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 24, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 122 illustrated in FIG. 22.

EXAMPLE 1 Illustrative

G-protein coupled receptors (e.g. serotonin, dopamine, glutamate,chemokine, and histamine receptors) are one class of proteins thatundergo a conformational change when activated by a ligand, and are thusamenable to study using the present invention. GPCRs that are notintrinsically nonlinear-active may be labeled using a nonlinear activelabel, and the conformation change is detected via a change in theorientation of the nonlinear active label. One or more labeled GPCRproteins can be attached to a surface, for example by embedding theprotein molecules in an array of supported lipid bilayer structures on aglass substrate, where each bilayer structure in the array contains asingle species of GPCR molecule. In a preferred embodiment of thepresent invention, each supported lipid bilayer structure is confinedwithin an individual well of a microplate. The conformation change thatresults when binding of a ligand activates a GPCR receptor causes achange in the orientation of the label with respect to the opticalinterface on which the molecules are immobilized, and thus a change inproperties of the nonlinear optical beams (e.g., second harmonic light)such as intensity, wavelength or polarization.

In a screening experiment, a microwell plate containing the immobilizedGPCRs is positioned on the translation stage mechanism of the highthroughput analysis system and moved into position for measuring asignal in a first well. A background signal can be measured for the oneor more GPCR samples prior to exposure of the immobilized GPCR moleculesto a test compound by measuring the nonlinear optical signal from thefirst well, repositioning the microwell plate to a second well to repeatthe background measurement, and so forth. Repeat measurements ofnonlinear optical signals are then made for each well at one (endpointassay mode) or more (kinetic measurement mode) defined time pointsfollowing the addition of the test compound, and analyzed to determineif the test compound induced conformational change in the one or moreGPCR species.

In some cases, binding of a test compound to a GPCR molecule may lead toa change in measured nonlinear optical properties even though the GPCRis not activated by the test compound. For example, this can be due toan interaction between the test compound and the GPCR molecule in thebound complex which alters the orientation of the attached label withrespect to the receptor molecule, rather than a change in theconformation of the receptor molecule. A control can be performed, ifdesired, to assign measured changes in nonlinear optical properties tobinding or activation of the receptor, for example, by using a compoundwhich is known to bind to a given GPCR receptor but not to produce aconformational change. If necessary, the position of the label on theGPCR can be altered by changing the conjugation chemistry of the labeland/or genetically modifying the receptor to introduce new labelingsites, in order to ensure that observed changes in nonlinear opticalsignal correlate to receptor activation or conformational change.

In the example described above, each of the GPCR molecules immobilizedin the array of lipid bilayer structures on the glass substrate (andfurther separated by means of the wells in which they reside) aresubjected to measurement and analysis by means of repositioning theglass substrate (microplate) with respect to the excitation light beamthrough the use of the precision X-Y translation stage, whilemaintaining efficient optical coupling via the recirculatingindex-matching fluid, index-matching elastomer, or prism grating designsdisclosed above.

Typically, the dispensing of test compound solutions into the wells ofthe microplate will be performed by a programmable, automatedliquid-dispensing unit that is integrated with the optical instrumentsystem. Each of the GPCR species immobilized in the wells of themicroplate may be exposed to the same test compound, or each may beexposed to a different test compound. In some aspects of the presentinvention, the high throughput analysis system will further compriserobotics for moving microwell plates comprising the GPCR samples to beanalyzed from an external storage system into a “home” position for thetranslation stage, or for replacing GPCR-containing microplates forwhich analysis has been completed with new sample plates. In otheraspects of the present invention, the high throughput analysis systemwill further comprise additional robotics for moving standardmicroplates containing the collection of test compounds into and out ofa “home” position for the liquid-dispensing unit.

In a preferred aspect of the present invention, a computer system (i.e.a processor or controller) is configured to run software for (i)controlling all programmable components of the high throughput analysissystem, (ii) synchronizing the operational steps of moving microwellplates into and out of position, taking background optical measurements,dispensing test compound solutions, and repeating the opticalmeasurements for use in end-point or kinetic mode assays, (iii) storingand processing the nonlinear optical signal data received from thedetector, and (iv) optimizing the overall throughput of analysis (e.g.in terms of number of GPCR sample/test compound combinations analyzedper hour) by using the input system setup parameters (e.g. number ofmicrowell plates to be analysed, number of wells per plate, number oftest compounds to be tested, endpoint versus kinetic measurement mode,etc.) to calculate an optimal order for interspersing backgroundmeasurement, test compound dispensing, and repeat measurement steps forthe different wells on each microplate.

EXAMPLE 2 Mold Design & Process for Fabricating Prism Arrays

A mold was created to fabricate the original “skip-prism” design shownin FIGS. 13A-13C, with the injection molding process performed by aspecialty optical molding house (Apollo Optical, Rochester, N.Y.). Theresulting parts show stress birefringence (see the crossed-polarizerimage in FIG. 14), and the measured SHG signal is adversely affected bythis birefringence (FIG. 15), with the signal dropping along the lengthof the prism array.

In order to reduce or eliminate the birefringence effect on SHG signal,we experimented with the type of plastic used (COP, COC, acrylic, etc),different molding processing conditions (temperature, pressure, flowrate, cycle time, etc), and post-molding annealing. We were able to findmaterials and process parameters that reduced the birefringencemodestly, but those conditions also tended to cause the plastic part toadhere tightly to the mold and fracture upon release from the mold.(Note: this mold contained ejection devices that pushed only on the ventand gate ends of the part). Annealing the part reduced stressbirefringence significantly, but caused excessive mechanical warping ofthe part thereby making it unusable.

Injection molding flow simulations indicated that stress was createdprimarily at the transition from the planar gate to the prism-structuredpart (results not shown). The simulation results also predicted that thepart could be made longer than required and then trimmed to length,thereby mechanically removing the stressed regions of the molded partand resulting in a prism array of the same final length as in theoriginal design.

The results of the mold flow simulation were used to design a new moldthat includes several significant changes:

-   -   1. The overall length of the part has been increased to allow        birefringent transition zones to be trimmed off.    -   2. Gate and vent features were made longer to reduce shear        stress in the plastic.    -   3. Mold eject features were added along the length and width of        the part to facilitate release from the mold and reduce the        chance of fracturing the part during release.    -   4. “Glue bumps” were added to the planar side of the array to        control the thickness of the glue layer when the prism array is        laminated to a glass-bottom microplate, and to improve glue        adhesion during temperature variation.

The new part design is shown in FIGS. 16A-16C, including the gate (left)and vent (right) features. FIG. 19 shows a cut-away version of the moldtool. Note that there is a 6×3 array of “ejector blade” devices (i.e.“ejection devices”) that are used during mold release to apply moreuniform pressure to the part during release (FIG. 20). Additionalejection features (not shown) are also used in the gate and ventregions. Generally, optical-quality molded parts do not use ejectorfeatures at all since ejectors impact on the surface of the part and cancreate blemishes. Since our prism array design has some regions that arenot optically addressed, we were able to arrange the ejector blades toimpact the part only in those regions where optical performance of thepart is non-critical.

In general, the larger the number of blade-like ejector features in anm×n array of ejection devices, the more uniform the pressure exerted onthe part during mold release. In some embodiments, m and/or n will havea value of at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20. In some embodiments, m and/or nwill have a value of at most 20, at most 19, at most 18, at most 17, atmost 16, at most 15, at most 14, at most 13, at most 12, at most 11, atmost 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most4, at most 3, or at most 2. The values of m and n may the same or may bedifferent, and may include any combination of values within theabove-specified range.

The new mold design, specifically the ejection features, allowed theinjection molding vendor to experiment with different pressure andtemperature profiles which would have caused release-fracture in theoriginal mold. These conditions effectively de-stress the plastic duringthe molding process, but cause the part to adhere more tightly to theoptical mold surface. The ejector features distributed across the lengthand width of the part allow the part to be ejected from the mold withoutfracturing. Prism parts made with new mold have negligible stressbirefringence, as shown in the cross-polarizer image in FIG. 17. SHGdata also shows a much more uniform signal response along the length ofthe prism array (FIG. 18). Residual birefringence is still detectable,but the extra length of the part allows us to optimize the trimlocations to cut off the transition regions where there is the mostbirefringence (data not shown).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method for determining interactions between target molecules andtest compounds, comprising: a) contacting at least one target moleculewith at least one test compound, wherein the at least one targetmolecule is labeled with a nonlinear-active label and is tethered to atleast one discrete region on an upper surface of a planar substrate; b)illuminating the at least one target molecule with one or moreexcitation light beams using total internal reflection of the one ormore excitation light beams from the upper surface of the planarsubstrate, wherein the planar substrate comprises an array of prismsintegrated with a lower surface, wherein the one or more excitationlight beams are directed by an entrance prism to a single discreteregion that is adjacent to but not directly above the entrance prism,wherein the one or more reflected excitation light beams and a nonlinearoptical signal generated by the at least one labeled target moleculetethered to the single discrete region are collected by an exit prismthat is also adjacent to but not directly underneath the single discreteregion, and wherein the entrance prism and the exit prism for the singlediscrete region are different, non-unique elements of the array ofprisms; and c) determining if a conformational change was induced in theat least one target molecule through contact with the at least one testcompound by detecting a change in a property of the nonlinear opticalsignal; wherein determinations of conformational change are performed atan average rate of at least 10 test entities tested per hour.
 2. Themethod of claim 1, wherein the determinations of conformational changeare performed at an average rate of at least 100 test compounds testedper hour.
 3. The method of claim 1, wherein the at least one targetmolecule is tethered to each of two or more discrete regions.
 4. Themethod of claim 1, wherein at least two different target molecules aretethered to at least two different discrete regions.
 5. The method ofclaim 1, wherein the target molecules are selected from the groupconsisting of cells, proteins, peptides, receptors, enzymes, antibodies,DNA, RNA, oligonucleotides, small molecules, and carbohydrates, or anycombination thereof.
 6. The method of claim 1, wherein the targetmolecules are drug targets or portions thereof.
 7. The method of claim1, wherein each of at least four different target molecules are tetheredto at least four different discrete regions.
 8. The method of claim 1,wherein each discrete region comprises an area of up to about 100 mm² onthe upper surface of the planar substrate.
 9. The method of claim 7,wherein each discrete region comprises a supported lipid bilayer. 10.The method of claim 9, wherein the target molecules are tethered to orembedded within the supported lipid bilayer.
 11. The method of claim 1,wherein the at least one test compound is selected from the groupconsisting of cells, proteins, peptides, receptors, enzymes, antibodies,DNA, RNA, oligonucleotides, small molecules, and carbohydrates, or anycombination thereof.
 12. The method of claim 1, wherein the testcompound is a drug candidate or portion thereof.
 13. The method of claim1, wherein the contacting step occurs in solution and comprisesutilizing a pre-programmed fluid dispensing unit to dispense the atleast one test compound.
 14. The method of claim 1, wherein thecontacting step comprises contacting each of at least four differenttarget molecules with a different test compound.
 15. The method of claim1, wherein the contacting step comprises serially contacting the atleast one target molecule with at least 100 different test compounds.16. The method of claim 1, wherein the contacting step comprisesserially contacting the at least one target molecule with at least10,000 different test compounds.
 17. The method of claim 1, wherein theone or more excitation light beams are provided by one or more lasers.18.-19. (canceled)
 20. The method of claim 1, wherein the non-linearoptical signal is selected from the group consisting of second harmoniclight, sum frequency light, and difference frequency light.
 21. Themethod of claim 1, further comprising moving said planar substraterelative to the position of one or more external sources of the one ormore excitation light beams.
 22. The method of claim 21, wherein eachdiscrete region of two or more discrete regions is optically coupledwith an entrance prism and a different exit prism that are bothintegrated with the lower surface of the substrate. 23.-24. (canceled)25. The method of claim 1, further comprising repeating the illuminatingand determining steps a plurality of times after said contacting step,thereby determining conformational changes in the at least one targetmolecule as a function of time. 26.-42. (canceled)
 43. The method ofclaim 1, wherein the change in a property of the nonlinear opticalsignal comprises a change in intensity, wavelength, or polarization.